This invention relates to genetic mutations and in particular to methods of generating and identifying genetic mutations in polyploid plant species.
In eukaryotic organisms, genetic information is encoded by DNA strands organized into sets of several chromosomes, or genomes, located within the cell nucleus. An organism containing a single copy of each chromosome set is referred to as a genetically monoploid organism. A convenient abbreviation for the monoploid complement of chromosomes is the letter xe2x80x9cnxe2x80x9d. For example, wheats have a basic monoploid complement of seven chromosomes, thus n=7. Most eukaryotic organisms, however, contain two copies of each member of the monoploid complement of chromosomes and are referred to as being genetically diploid (2n), with each chromosome existing as a member of a homologous pair of chromosomes. In some organisms, the diploid genome (i.e., the sum total of the genetic information encoded by the diploid number of chromosomes) is further duplicated to yield a chromosome complement consisting of multiple copies of the monoploid set of chromosomes.
Polyploid, is the generic term for an organism having more than the diploid number of chromosome sets, or genomes. Polyploidy is predominantly, although not exclusively, found in plants, especially within the agriculturally important cereal species, such as wheat and oats. Over the course of agricultural history, numerous polyploid varieties of crop species have evolved, possibly because of the improved vigor, larger grain or plant size often associated with polyploidy. Polyploidy may naturally arise by the spontaneous duplication of one or more genomes (autopolyploidy), or by the much more common process of genetically combining two or more genomes, or complete sets of chromosomes, from genetically different parents (allopolyploidy). For example, the spontaneous, natural doubling of the chromosome set of a diploid (2n) species, results in the creation of a novel autotetraploid (4n) species. The two diploid (2n) genomes that constitute the autotetraploid (4n) genome are referred to as homologous genomes, because they are genetically identical, having arisen by the duplication of a single diploid genome.
However, the true nature of autotetraploids is more complicated than appears, because true autoploids, whether spontaneous or artificially induced, are rarely fully fertile or genetically stable. All apparent autotetraploid species are only reproducible because their genomes have to some degree diverged, even though some pairs of genes show tetrasomic inheritance. A cross between two genetically divergent diploid (2n) species, in which reduction division during meiosis fails to occur, results in an allotetraploid (4n) species. In the case of the allotetraploid, however, the two diploid (2n) genomes that constitute the allotetraploid (4n) genome, are referred to as homoeologous genomes, because although they are genetically very similar, they are not genetically identical, having arisen by the fusion of two, comparatively different, independently evolved, diploid genomes.
The genetic information on the DNA strands of the chromosomes of all organisms is located in discrete segments of the chromosome DNA, termed genes. All genetic differences among natural (or artificial) species and varieties, results from mutational modifications in the structure and function of the genes. Such structural gene modifications in natural species and varieties are considered to have occurred spontaneously. The probable basis for such modifications is unknown, but there is evidence indicating that errors in DNA synthesis do infrequently occur, perhaps initiated by a wide variety of environmental and nutritional conditions. Mutations are important, in that they form the entire genetic basis for the evolution of species in nature and the basis for the artificial development of new plant cultivars. If enough different mutational variations are accumulated, the mutations form the basis for the development of new sub-species and species variations in all organisms, not only in plants.
In more modern times, geneticists and plant breeders have used mutation induction technology to supplement or complement the naturally-occurring genetic variations to improve numerous characteristics or properties of plants. Mutagen treatment technology has evolved over a long period of years, and only in the past 10 years or so has mutation induction, as a method to improve plants, become a technology of increasing acceptance. Although some methods for mutagen application have been described in the literature, and are useful, new techniques have been developed more recently that are significantly more effective and efficient in terms of resources. The mutagenesis technology embodied in this invention, represents an advance from the methods described in available literature (IAEA Manual on Mutation Breeding-Tech. Reports Series 119, IAEA, Vienna, 1977). Mutagenesis technologies in use for plant genetics and breeding research today, especially for small grain cereals mostly involve applications of the mutagens to seeds. The most widely used mutagens include electromagnetic radiations, X-rays and gamma rays, and nuclear radiations, such as thermal or fast neutrons, mainly because the sources of these radiations are more available. More commonly, chemical mutagens are now used in research; the preferred chemical agents are such alkylating agents as ethyl methanesulfonate (EMS), and diethyl sulfate (DES). In addition, azide in the form of sodium or potassium azide is now widely used. Less commonly employed for mutation induction are the more hazardous carcinogenic agents, such as N-methyl nitrosourea and N-ethyl nitrosourea, and the highly carcinogenic nitrogen mustard, 2-chloroethyl-dimethylamine. The nitrosoureas are especially active mutagens (Maluszinski, M. Acta. Soc. Bot. Pol. 51:429-440, 1982) whereas use of the nitrogen mustards poses a significant health risk to the user because these compounds are highly toxic to humans. More recently, as cell (microspore) and tissue culture research have evolved, attention is being given to the application of mutagens to accelerate the frequency of mutations regenerable from such cultures. However, mutagenesis technology in cultures is still in its developmental infancy, as are applications of the technology for plant improvement.
Most applications of mutagens to produce useful variants in crop plant species have had a primary goal, such as reducing plant height, reducing grain shattering, or changing the photoperiod response, traits for which the genetic basis in the mutagenized variety was previously unknown. Even so, unexpected, as well as expected results have been achieved from many studies. As a result, many new cultivars of crop plants have been developed via the direct release of a genetic line differing from the original genotype by an induced mutation (Konzak, C. F., Role of Induced Mutations, 1983, pp. 216-292. In: Crop Breeding-a Contemporary Basis. P. B. Vose and S. G. Blixt (eds). Pergamon Press, Oxford and New York; Micke, A. and Donini, B., 1987, Tropic. Agric. (Trinidad) 64:259-277). An even larger number of new cultivars of crop plants have been developed using induced mutations as new genetic variability, demonstrating that induced mutations not only can be useful, but also that they can be used in breeding to advance the potential yield, quality or disease resistance, of many crops (Micke, A. and Donini, B., 1987, Tropic. Agric. (Trinidad) 64:259-277). However, there remains a need for a method of inducing a wide range of mutations that is generally applicable to crop plants. Typically, mutagens have been applied to seeds of various species to induce mutations that might be expected to occur, based on an expectation that such genetic variation should be inducible. In most of the examples described in the literature, the actual numbers of mutations of a general phenotype generally have been sufficiently high for other scientists to recognize them as being induced, especially the relatively common mutations of semi-dwarf, or reduced height phenotypes. But, even among these more frequent types of mutations, those at the same gene locus have been rarely isolated in the same experiment. Thus, the primary evidence that the new phenotypic/genotypic variants are induced mutations, has largely been assumed because of the simultaneous recovery of many other mutant phenotypes in the same study. Studies to induce white seed coat color in wheat, for example, have been successfully carried out in red seed coat wheats, without knowledge of the genetic structure of the mutagenized wheat genotype for genes controlling the seed coat color, though seed coat color in wheat is known to be controlled by three homoeologous gene pairs, located in the A, B and D genomes. In recent, unpublished work, Warner isolated 3 white-seeded xe2x80x98mutantsxe2x80x99 in Chinese Spring wheat, a genotype widely known to carry only 1 dominant red seed coat color gene. The three xe2x80x9cmutantsxe2x80x9d were essentially identical, hence may have originated from a single event, for which the frequency is sufficiently low that a spontaneous origin cannot be ruled out.
There is a continuing need to identify new mutants, having novel, desirable characteristics, within agriculturally important plant species. Novel mutations might convey for example, increased resistance to drought or cold, reduced plant height, non-shattering of grain, resistance to preharvest sprouting, as well as new, or modified quality characteristics, offering new market use opportunities, or might result in higher crop yields. Further, it is desirable to generate numerous mutations within a plant species in order to obtain novel phenotypes, which can be intercrossed to develop novel plant cultivars having defined, more desirable characteristics of economic value. The process of identifying novel mutations within polyploid species is complicated, however, by the fact that most mutations are recessive, i.e., the mutant phenotype is not apparent in the presence of one or more copies of the non-mutated, dominant gene. Thus, the phenotype of most mutants will not be apparent in a polyploid species unless all copies of the relevant gene bear the same mutation. Since polyploids, as the term is used herein, generally contain two or more pairs of each gene, the probability of fortuitously generating an individual plant having all copies of a gene mutated in a similar manner is very slight. Thus, there is a need for a method that is generally applicable to polyploid plant species, which permits the creation and selection of a large number of mutations in any desired, target gene.
The present invention provides methods for generating and identifying mutations in any target gene of a polyploid plant species. In one aspect of the present invention, a plant is selected that has at least one pair of functional, target genes located exclusively in only one of its homoeologous, or homologous, genomes. Seed derived from the selected plant are then contacted with an effective amount of at least one mutagenic agent, the treated seed are germinated and the seeds or plants derived therefrom, are screened for mutations in the target gene. In a presently preferred embodiment of the present invention, the selected plant is a cereal crop plant and the mutagenic agent is a sequentially applied combination of ethyl methane sulfonate followed by sodium azide. In another aspect of the present invention, selective matings are made to construct plant genotypes with a functional target gene pair exclusively in only one of their homoeologous, or homologous, genomes. Seed derived from the constructed plants are then contacted with an effective amount of at least one mutagenic agent, the treated seed are germinated and the seed or plants derived therefrom, are screened for mutations in the target gene. In a presently preferred embodiment of this aspect of the present invention, the constructed plant is a cereal crop plant and the mutagenic agent is a sequentially-applied combination of ethyl methane sulfonate followed by sodium azide. In yet another aspect of the present invention, polyploid wheat plants, mutated in accordance with the methods of the present invention, are provided that include mutations in all copies of the waxy gene and so synthesize starch that has a reduced amount of, or completely lacks, amylose. Thus, the inventive concepts set forth herein can be used to create, select and identify mutations in any target gene of any suitable polyploid plant. The mutations generated in accordance with the present invention provide a source of numerous, readily-identifiable mutations that can, if so desired, be used as germplasm to generate novel new plant cultivars, or the novel induced mutant alleles in different genomes of the polyploid, can intercrossed to generate novel phenotypes having predetermined, desirable properties.